DESCRIPTION: We seek to develop and evaluate novel biomaterial/growth factor therapies to heal diabetic foot ulcers, which plague diabetes patients as a major diabetes complication, resulting in substantial morbidity including amputation and reduction of lifespan and quality of life. In current clinical practice, growth factor therapies have generally been a disappointment: Based on our published and unpublished preliminary work, we believe that the major problem with growth factor therapeutics in general is that the evolutionary context of the growth factor-extracellular matrix (ECM) microenvironment has been ignored. Our group has elucidated complex biomolecular interactions between growth factors and proteins of the ECM, which dramatically alter the biology of the growth factor. Indeed, we have come to believe that most growth factors evolved to function in concert with an ECM protein (Martino et al., Science Transl. Med. 2011, among other references). Here, we will evaluate both growth factor engineering and ECM engineering approaches directed to control the dynamic reciprocity between growth factors and the ECM in diabetic wound healing. We will on the one hand engineer growth factor variants to bind with super-affinity to the ECM, including fibrin, our selected translational biomaterial platform for ECM engineering; and we will engineer the ECM to bind with high affinity and promiscuity to growth factors. We will work in a mouse type 1 diabetes (T1D) model of delayed healing to explore responses relevant to diabetic foot ulcers, establishing a mouse clinic in which animals are treated with insulin so as to control hyperglycemia yet not restore normal euglycemia, to model imperfect glycemic control in patients. (Specific Aim 1) We will employ a protein engineering approach to engineer ECM super-affinity variants of VEGF-A, PDGF-BB, HB-EGF and CXCL12 in the NOD mouse model of delayed wound healing, targeting VEGF-A for its ability to induce angiogenesis, PDGF-BB and HB-EGF for their ability to induce re- epithelialization, and CXCL-12 for its ability to induce progenitor cell infiltration. Specifically, based on our preliminary work (Martino et al., Science 2014), a high affinity and promiscuous ECM binding domain from PlGF-2 (PlGF-2123-144) will be fused to these factors, to yield VEGF-A-PlGF-2123-144, PDGF-BB-PlGF-2123-144, HB-EGF-PlGF-2123-144 and CXCL12-PlGF-2123-144. We present preliminary data with combined VEGF-A-PlGF- 2123-144, PDGF-BB-PlGF-2123-144, dual factor therapy in a murine T2D model of diabetic wounds. (Specific Aim 2) We will do this in fibrin stabilized with a novel protease inhibitor, a human protein to replace the bovine aprotinin currently used in fibrin sealants, to avoid immunological complication upon repeated use. (Specific Aim 3) Finally, we will utilize a high affinity promiscuous growth factor binding domain that we have discovered (unpublished) in von Willebrand factor (vWF1328-1351) bound in fibrin to create high affinity binding sites to both native and engineered growth factors. Our objective is to produce a stabilized fibrin matrix, with one or perhaps two bioactive factors, to heal wounds in T1D patients.